Potassium (K+) channels are major determinants of cell excitability and play crucial roles in many physiological processes. There are two major mechanistic features of K+ channels: 1) permeation, which comprises both the magnitude of ionic flow through their pores and the selectivity of the pore (acute discrimination between ion types), and 2) gating, the process of opening and closing the pore to ionic flow. Malfunction in either of the above can have disastrous physiological consequences. The overall objective of this grant is to understand mechanisms of ion conduction, selectivity and gating in K+ channels by employing structure-based functional analysis. To accomplish this goal, model bacterial K+ channels, homologues of the eukaryotic K+ channels involved in excitability, will be used. Bacterial channels lend themselves more readily to the biochemical and structural studies necessary to investigate these mechanisms in detail. Our first major aim is to understand aspects of permeation in potassium channels. Aim 1.1 will investigate KcsA selectivity using a multidisciplinary three-pronged approach: 1) electrophysiology of KcsA block by impermeant ions using analysis of single- channel current recordings, 2) X-ray crystallography of the blocked KcsA channels, and 3) molecular dynamics to calculate energetic landscapes as ions negotiate the KcsA pore. Although several different mechanisms have already been proposed for K+ channel selectivity, our emerging functional, structural and molecular dynamics studies may lead towards a novel picture. Aim 1.2 will identify the molecular mechanism for uneven conduction through two structurally similar potassium channel pores. The role that negatively charged amino acids, located by the extracellular pore mouths of both MthK and KcsA, play in tuning ion conduction using electrophysiological and X-ray crystallographic analysis of KcsA and MthK mutants, will be investigated. Our second major aim investigates aspects of gating in K+ channels. Aim 2.1 will probe the mechanism of proton activation in KcsA. Analysis of the functional and thermodynamic consequences of single and pair-wise mutation of residues at the channel bundle crossing will elucidate which residues directly interact or couple to open the channel with protons. In order to gain further insight into the mechanism, the structures of KcsA mutants with altered gating will be crystallized and solved. Aim 2.2 will investigate the location of the MthK channel activation gate by obtaining structures with X-ray crystallography of wildtype and mutated MthK in the closed conformation, and by determining whether the access of intracellular blockers to the channel pore is dependent on whether the channel is open or closed. Channel block for MthK will be compared to similar experiments for KcsA, a channel known to gate with a bundle-crossing constriction. The proposed aims will provide important new insights into the fundamental nature of potassium channel properties, and much of the information may be readily applicable to our understanding of the broader family of ion channels.